Twenty-five healthy individuals and 34 ADHD patients between 9 and 16 years participated in this event-related potential (ERP) study. Healthy controls underwent single task versions of a 2-back working memory task and a go/nogo response inhibition task as well as the introduced combined 2-back/nogo task. This combined task demonstrated a comparable ERP structure for working memory and response inhibition aspects as single task versions. Behaviorally, higher working memory performance during the combined paradigm indicated lower task difficulty, while high correlations between combined and single task versions still indicated valid working memory measures. For response inhibition performance, different task versions resulted in similar outcomes. The application of the combined n-back/nogo paradigm in ADHD patients revealed the expected working memory and response inhibition deficits, increased omission errors, reaction times, and standard deviation of reaction time, as well as diminished n-back P3 and nogo P3 amplitudes. We conclude that the combined n-back/nogo task is an effective paradigm for the economical assessment of working memory and response inhibition deficits in ADHD on a behavioral and neurophysiological level.The development of cognitive interventions in attention-deficit/hyperactivity disorder (ADHD) often requires the assessment of multiple cognitive functions. However, experimental settings consisting of various tasks are particularly strenuous for patients and can thus result in poor data quality. For the economical assessment of working memory and response inhibition, this study aims to validate a combined  Attention-deficit/hyperactivity disorder (ADHD) is one of the most frequent disorders in child and adolescent psychiatry, with a worldwide prevalence of about 3.4% . AdditioTherapeutic approaches that aim for the improvement of cognitive functions in ADHD include cognitive training , neurofen-back task. During this task, participants decide for a series of stimuli if the current stimulus is identical to the one presented a specified number n of trials earlier. With increasing n and therefore increasing working memory load, the task becomes more demanding. There is strong evidence that ADHD patients show impaired performance as well as higher reaction time and standard deviations of reaction time during n-back tasks  during the application of single and combined task versions. EEG analysis is optimally suited to investigate neural mechanisms underlying behavioral data. For that purpose, we focused on the following event-related potential (ERP) components as they represent the executive processes that show pathological changes in ADHD . Trained psychologists diagnosed participants on the basis of clinical interviews, which were conducted with all participants and their parents using the German Adaptation (p ≥ 0.085) but in subjective and objective assessment of attentional impairments .Fifty-nine participants aged between nine and 16 years were recruited via the Department of Child and Adolescent Psychiatry and through advertisements in a local newspaper. Thirty-four were diagnosed with ADHD according to DSM-5 criteria . Patients with comorbid psychiatric or neurologic diagnoses were excluded from the study. This also applied to dissocial disorders, because differential ERP patterns were demonstrated between patients with ADHD only and patients with comorbid diagnoses as oppositional defiant disorder or conduct disorder . Eleven aptation  of the Saptation . As suppaptation  and behaaptation , and addaptation . IQ valuaptation . Last, taptation  was applThe study followed the ethical standards of the Declaration of Helsinki and was approved by the local ethics committee of the Otto-von-Guericke University Magdeburg. All caregivers and participants gave their written informed consent and assent, respectively. Participants were reimbursed with a voucher of 15–20€ for a local shopping center.n-back and go/nogo task versions as well as the combined n-back/nogo task within one session. The order in which cognitive tasks were applied was pseudo-randomized between participants, with balanced frequency for each possible sequence. ADHD patients underwent only the combined n-back/nogo task. Task illustrations are provided in ® (version 18.0)1. Stimuli had a visual angle of 0.86° (height) and were presented in black on a gray background . In all tasks, stimulus duration was 500 ms and participants had 2000 ms to give their response. The interstimulus interval was 2500 ms. The paradigms that were used in this study can be found under https://doi.org/10.6084/m9.figshare.c.4933326.The healthy control group performed single n = 2). These target trials had a proportion of 22%. If stimuli were identical, participants pressed a button with their right hand, and if stimuli were not identical, they pressed a button with their left hand. The key assignment was the same for all participants. The task consisted of three runs that had a duration of 3.8 min each  and that were separated by pauses of at least 30 s. The task started with a training run of 2.5 min  with feedback indicating right or wrong reactions. After this training, the investigator decided if the participant understood the task correctly or if the training must be repeated.A series of capital letters was presented  and participants decided if the current stimulus was identical to the stimulus two trials earlier . The task consisted of three runs with a duration of 4.6 min each . It started with a short training run of 40 s  with feedback of right or wrong reactions, which could be repeated if required.n-back sequences containing one to eleven trials , with the last trial of each sequence always being a nogo trial. Participants were informed that there was never an n-back target trial directly after a nogo trial. The task was split into three runs with a duration of 4.6 min each , providing an equal quantity of n-back target trials to the single n-back task. It started with a training run of 2 min  with feedback of right or wrong reactions, which could be repeated if required.Again, letters were presented sequentially and participants decided if the present and the 2-back stimulus were identical (21%). Additionally, participants were instructed to withhold their response when the letter X appeared. Those nogo trials had a proportion of 17%. The task was composed of a random order of n-back/nogo task in the initial of several sessions for four runs while EEG was recorded (n = 24). The other was an application-oriented study, where the task was applied in one of three sessions for six runs during a placebo non-invasive brain stimulation while EEG was recorded during the last three runs (n = 10)  . Data frEEG was recorded with a SynAmps amplifier  from 21 channels. For this purpose, Ag/AgCl-electrodes were placed in an EEG cap , according to the International 10–20 EEG system . The ground electrode was placed at position AFz, and the EEG was referenced to linked mastoids. Via two bipolar channels, EOG was recorded with electrodes placed at the outer canthi of both eyes and at sub- and supra-orbital positions. Impedances were kept below 15 kΩ, and data were recorded with a sampling rate of 500 Hz. A high pass filter of 0.05 Hz, a low pass filter of 70 Hz, and a notch filter of 50 Hz were applied online.n-back target trials and three from analyses of nogo trials. In the control group, 34.3 (SD = 7.7) n-back targets and 39.3 (SD = 6.7) nogo trials were analyzed on average in each participant for single task versions. A mean number of 48.1 (SD = 8.5) n-back targets and 42.0 (SD = 6.9) nogo trials of the combined n-back/nogo task remained in the analysis of controls. In ADHD patients, a mean of 38.4 (SD = 19.8) n-back targets and of 41.3 (SD = 14.7) nogo trials were analyzed on average.We analyzed data with EEGLAB  and ERPLn-back P3 analysis was 275–500 ms in both, single n-back and combined n-back/nogo tasks. The nogo N2 component was analyzed at 225–350 ms in the single go/nogo task and at 250–500 ms in the combined n-back/nogo task. For P3 analysis in the single go/nogo task, a time window of 325–600 ms was used for nogo trials and of 225–425 ms for go trials. In the combined n-back/nogo task, the nogo P3 was analyzed at 425–625 ms. The n-back P3 component has a centro-parietal maximum  with higher values indicating better performance. Response inhibition performance was the inverse of nogo commission error rate , thus better performance was indicated by higher values. The analyzed behavioral measures were working memory performance, response inhibition performance, omission errors, reaction time, and standard deviation of reaction times. If Levene’s test for variance equality was significant, Satterthwaite approximations for the degrees of freedom are reported for t-tests. ERP components were characterized by peak amplitudes and latencies. These were analyzed using ANOVAs while significant main and interaction effects were further investigated via post hoc pairwise comparisons with Bonferroni adjusted alpha levels. We applied Greenhouse–Geisser corrections if assumptions of sphericity were violated. Effect sizes are reported as Cohens d for t-tests and as η2 for ANOVAs.Statistical analyses were conducted with SPSS . We compared sample characteristics between the control and ADHD group using independent samples n-back/nogo task using paired-samples t-tests. Reaction time distributions were characterized and inverse efficiency scores (IES) were determined. The IES is a measure of the speed-accuracy tradeoff, which is defined as the mean reaction time divided by the proportion of correct responses , Region  and Hemisphere (left vs. midline vs. right) were conducted for ERP characteristics. Data for the different levels of Region and Hemisphere were averaged across the following electrodes: frontal – F3, Fz, F4; central – C3, Cz, C4; parietal – P3, Pz, P4; left – F3, C3, P3; midline – Fz, Cz, Pz; right – F4, C4, P4. Second, we tested if components differed between each single task version and the combined n-back/nogo task, using repeated measures ANOVAs with the factors Task (single vs. combined), Region, and Hemisphere. Only main and interaction effects including the factor Task were reported for this analysis.For ERP analysis, first, we characterized n-back/nogo task were compared between the control and ADHD group using independent-samples t-tests. Further, the Pearson’s r correlation coefficient was calculated between working memory and response inhibition performance within the combined n-back/nogo task. ERP components were analyzed in mixed ANOVAs including the between-subjects factor Group (controls vs. ADHD) and within-subjects factors Region and Hemisphere. For this analysis, only main and interaction effects including the factor Group were reported.Behavioral measures during the combined Post hoc power analyses were conducted using the software G∗Power version 3.1  = 6.91, p < 0.001, d = 1.11]. Response inhibition performance was not significantly different between task versions  Further, we found a borderline significant trend toward a reduced number of omission errors in the combined n-back/nogo task compared to the single n-back task . Reaction times and standard deviations of reaction times for the combined n-back/nogo task were significantly higher than for go stimuli of the single go/nogo task  and were rather comparable to the single n-back task . Comparisons between task versions indicated better performance for the combined n-back/nogo task versions, reaction time distributions were characterized further (n-back/nogo task (excess kurtosis = −1.23) and was thus, more similar to the reaction time distribution of the single n-back task (excess kurtosis = −0.66). Further, while the reaction time distribution of the single go/nogo task was moderately skewed left (skewness = 0.94), reaction time distributions were approximately symmetric for the single n-back task (skewness = 0.33) and for the combined n-back/nogo task (skewness = 0.27). Moreover, regarding the IES we found no significant task difference for working memory performance , but for response inhibition the IES was higher in the combined n-back/nogo task (820 ms) compared to the single go/nogo task (480 ms), .As reaction time measures differed significantly between single go/nogo and combined  further . While tr correlation coefficients of behavioral measures between task versions revealed that working memory performance was highly correlated between the combined n-back/nogo task and the single n-back task . Further, the number of omission errors in the combined n-back/nogo task was correlated to omission errors in the single n-back task  as well as in the single go/nogo task . Reaction times in the combined n-back/nogo task were correlated to reaction times in both single task versions  and the same applied for standard deviations of reaction times . For response inhibition performance, we found no significant correlation between task versions . However, data inspection revealed two outliers for this correlation . However, the same participants as above were identified as outliers and excluded for an exploratory analysis, resulting in no significant correlation . In the combined n-back/nogo task, working memory and response inhibition performance were not significantly correlated with each other .Pearson’s  compare . When thn-back targets and non-targets during the single n-back task revealed main effects of Stimulus , Region , Hemisphere , and an interaction between Stimulus and Hemisphere . This analysis indicated larger P3 components in response to target than to non-target stimuli and at parietal than at central electrode positions. Post hoc tests showed larger amplitudes at midline compared to left electrode sites for target and non-target stimuli  while only for non-targets amplitudes were larger at right compared to left sites (p = 0.011). P3 amplitudes did not significantly differ between single n-back and combined n-back/nogo tasks .The comparison of P3 amplitudes between n-back P3 between target and non-target trials in the single task version resulted in an interaction effect between Stimulus and Region , indicating higher latencies at central than parietal positions during non-target trials (p = 0.007). A further interaction between Stimulus and Hemisphere was found , but post hoc tests did not reach significance. Subsequent analyses showed no latency differences between single and combined task versions .Latency comparisons of F = 7.59, p = 0.011, η2 = 0.240] and Region , as well as an interaction between Region and Hemisphere . Hence, N2 peaks were more negative for nogo than for go stimuli and at frontal than at central electrode positions. Focusing on frontal sites, amplitudes were more negative at the midline compared to the right electrode (p < 0.001) whereas there was no difference between central sites . Task version did not affect N2 amplitudes .The comparison of N2 amplitudes between go and nogo stimuli of the single go/nogo task, showed main effects of Stimulus , at frontal than at central electrode sites , and they were highest at right sites . Latencies were increased during the combined n-back/nogo task in comparison to the single go/nogo task .During the single go/nogo task, N2 latencies were higher for nogo than for go stimuli , Region , and Hemisphere , interactions between Stimulus and Region , Stimulus and Hemisphere , Region and Hemisphere , and a threefold interaction . Nogo stimuli elicited larger P3 components than go stimuli. This effect was greatest at central electrode positions, but was significant also at frontal and parietal sites . Generally, P3 amplitudes were largest at parietal and smallest at frontal positions . However, during nogo trials, P3 amplitudes were centro-parietally largest at midline sites  while during go trials P3 amplitudes were frontally, largest at the right site . The next comparison between single and combined task versions revealed interactions between Task and Region  as well as between Task, Region, and Hemisphere , indicating larger P3 amplitudes during the combined n-back/nogo task at midline and right parietal electrode positions .Comparing P3 amplitudes between stimulus types of the go/nogo task, we found main effects of Stimulus . Further, latencies were increased in the combined n-back/nogo task compared to the single go/nogo task .We found higher P3 latencies during nogo than during go trials , and response inhibition performance was reduced to 77.85% . Moreover, patients showed more omission errors  as well as higher reaction times  and higher standard deviations of reaction times . Details of all comparisons are given in As expected, group comparisons revealed that ADHD patients showed deficits in all behavioral measures during the combined n-back/nogo task . This was not associated with reported attention problems  or attentional performance  .In ADHD patients, we found a significant correlation between working memory and response inhibition performance assessed with the combined n-back P3 component were found  but latency was not significantly different between the control and the ADHD group .In ADHD patients, diminished amplitudes of the F = 0.95, p = 0.335, η2 = 0.017]. However, ADHD patients showed delayed N2 latencies .Amplitudes of the nogo N2 component did not differ between groups  indicating a reduced peak amplitude in the ADHD group. Moreover, an interaction between Group and Hemisphere was revealed . This indicated that controls showed largest P3 peaks at midline electrode positions , while in patients differences between midline and lateral electrodes were reduced but still significant . Further, in controls P3 peaks were larger at right compared to left electrode sites (p = 0.050) but this effect was missing in ADHD. The group analysis revealed no latency differences of the nogo P3 component .A main effect of Group was identified for the nogo P3 amplitude [https://doi.org/10.6084/m9.figshare.c.4933326.ERP data are illustrated in n-back/nogo paradigm that combines working memory and response inhibition aspects and hypothesized that its behavioral and ERP characteristics would be comparable to those of parallel single task versions. Contrary to our expectations, working memory performance was higher in the combined n-back/nogo task than in the single n-back task but still, both measures were highly correlated. We confirmed that response inhibition performance was similar between the combined n-back/nogo and the single go/nogo task version. Further, we found that reaction times and standard deviations of reaction times in the combined n-back/nogo task were comparable to those of the single n-back task but were higher than those of the single go/nogo task. As expected, the combined n-back/nogo paradigm demonstrated comparable ERP structures as single task versions for working memory and response inhibition task aspects. Still, we found larger nogo P3 amplitudes as well as increased nogo N2 and nogo P3 latencies for the combined task. The application of the combined paradigm in ADHD patients revealed the expected working memory and response inhibition deficits, increased omission errors, reaction times, and standard deviations of reaction time, as well as diminished n-back P3 and nogo P3 amplitudes. However, we found no reduction of the nogo N2 amplitude in patients.In the present study, we introduced the n-back/nogo task compared to the single n-back task. Despite this difference in performance outcome, we argue that the combined task delivers a valid measurement of working memory, because working memory performance was highly correlated between combined and single task versions. Therefore, the improved performance outcome seems to result from a reduced difficulty of the combined paradigm. This is remarkable, as previous studies found that working memory performance decreased when inhibitory task aspects were added . The names of the repository/repositories and accession number(s) can be found in the article/The datasets presented in this study can be found in online repositories (The studies involving human participants were reviewed and approved by the Ethics Committee, Otto von Guericke University Magdeburg. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.CB-Z and KK conceived of the idea in the present study. KK and JT established the diagnostic procedure of participants. CB-Z and JT developed the paradigms. CB-Z implemented the tasks, carried out most of the experiments, and performed the data analysis. CB-Z and KK shaped the analysis. All the authors contributed to the interpretation of the data. CB-Z prepared the manuscript. JT, H-HF, and KK contributed to the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The subunit ligation is triggered by the heterodimerization of an arginine rich leucine zipper motif with a glutamic acid rich leucine zipper motif. The one-by-one ligation of hetrosubunits constructs the pairing enzymes RTA&AlaDH and AlaDH&FDH. The ligation modes were analyzed based on blue native polyacrylamide gel electrophoresis (BN-PAGE). The spectra of circular dichroism (CD), fluorescence, and two-dimensional FTIR provide information on the secondary structures and stability of the pairing enzymes. The enzyme-substrate interaction was analyzed based on microscale thermophoresis analysis. In contrast to the mixed three enzymes RTA + AlaDH + FDH, the ligated enzymes RTA&AlaDH + AlaDH&FDH exhibited a much larger substrate affinity, higher stability, and significantly enhanced activity.For the multimeric enzymes R-ω-transaminase (RTA), alanine dehydrogenase (AlaDH), and formate dehydrogenase (FDH), peptide bond formation between the hetrosubunits has been achieved by the intein-mediated  In this approach, alanine is used as amine donor. The coproduct pyruvate is recycled back to alanine by consuming ammonia and formate  to cooperate with  formate   (E)   and a gl (R) (E)  to triggEscherichia coli strain DH5α used as a host for DNA manipulation and strain BL21 (DE3) as a host for expressing enzymes were both purchased from Zoman Bio. And all other reagents were purchased from Sigma-Aldrich .DNA ligase, DNA polymerase, and restriction enzymes were purchased from Thermo Fisher Scientific. Oligonucleotide primers were synthesized by BioMed Tech. Archaeoglobus fulgidus (EC:1.4.1.1)  gene was synthesized based on the genome sequence of 1.4.1.1)  and clonAlaDH-rp , at the AlaDH-rp . The ZE- primers . The purR-ω-transaminase (RTA) gene used here was the same as that in our previous work  cells. The cells were cultured in Luria-Bertani medium (50 μg/ml ampicillin) under shaking (220 rpm) at 37°C for 3 h. At this time, the OD600 reached about 0.4, and the cells grew at exponential phase. Then the cells were induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), and the culture temperature was changed to 25°C. At this temperature the cells grew for 8, 12, and 16 h, respectively. During the co-expression of the precursors ZE-IntC-link-RTA and AlaDH-link-IntN-ZR, the in vivo subunit splicing occurred simultaneously. Both SDS-PAGE and blue native polyacrylamide gel electrophoresis (BN-PAGE) were used to monitor and analyze the crude proteins released from the cells. It was found that 8 h is enough for the cell growth to accomplish the in vivo subunit splicing, determined by observing the bands for the splicing products.The genes were transferred into E. coli cells were collected after 30 min centrifugation at 7000 g at 4°C. The cells were then disrupted under ultrasonication on ice. The cell debris was removed by centrifugation at 7,000 g at 4°C for 30 min. The supernatant was collected into a fresh tube. The enzymes were first purified by passing through a chromatography column (GE Life Sciences) filled with Ni Sepharose beads (GE Life Sciences). Five hundred mM imidazole was used as the elution liquid, which was separated from the purified enzymes by dialysis into a buffer . The purified enzymes were further fractionated by ultrafiltration . The splicing products were finally purified using a gel filtration chromatography .The 2. After dialysis, RTA&AlaDH (200 nM) was mixed with the ligand 2-octanone (100 mM), and then incubated at 30°C for an hour. A 16 step dilution series from 10 mM to 0.25 μM were prepared by DMSO. 10 μL of each step was filled into 16 fresh small micro reaction tubes and was well mixed by pipetting up and down. The measurements were performed at 20% IR-laser power and 40% MST power. Using the software MO. Affinity Analysis, the binding coefficient Kd values were obtained by fitting the fraction of bound protein to the quadratic solution of the binding reaction equilibrium. MST benefits from very low sample consumption and short measurement times.The experiments were performed on an instrument of microscale thermophoresis (MST) (Nano Temper technologies). The splicing product RTA&AlaDH was not labeled by fluorescence. The dialysis liquid consisted of 50 mM Tris-Cl (PH 8.0), 150 mM NaCl, and 10 mM MgCl–1. Each spectrum is the result of the accumulation and averaging of 512 interferograms. Ultrapure nitrogen was continuously introduced into the sample compartment to minimize the spectral contribution of atmospheric water. The enzyme was dissolved in 10 mM deuterated potassium phosphate buffer (pH 7.4) with a concentration of 15 mg/mL. The enzyme solution was rapidly prepared at 10°C, thus condensation of atmospheric water into the solution can be avoided. D2O was added into the sample of the enzyme solution, and the measurement of FTIR spectra was started. The hydrogen-deuterium exchange experiment was carried out for 90 min. The pure spectrum of enzyme was obtained by subtracting the spectra of D2O.FTIR- attenuated total reflection (ATR) spectra were recorded on a spectrometer (Bruker Tensor 27) at a nominal resolution of 2 cm2HPO4 and 100 mM NaH2PO4 in proportion to pH 8.0), then alanine (5 mM), pyridoxal-5′-phosphate (1 mM), 1 mM NAD+, and 100 mM ammonium formate were added. The reaction volume was 2 mL. The chromatographic pure methanol and ultra pure water were respectively filtered by the filter membrane, and then the ultrasonic treatment was carried out to exhaust bubbles for half an hour. The concentration of the substrate 2-octanone was monitored by HPLC (Shimadzu LC-10A) with ultraviolet detector at 280 nm using a C18 column . Setup column thermostat at 30°C, and the mobile phase was methanol/water (60:40 by vol) at 0.8 mL/min, and the injected sample was 20 μL.For transformation of the amines, the enzymatic catalysis was performed at 30°C under shaking at 200 rpm. The enzyme concentrations were: 0.5 mg/mL RTA&AlaDH + 0.5 mg/mL AlaDH&FDH; 0.5 mg/mL RTA#AlaDH + 0.5 mg/mL AlaDH#FDH. The concentration of substrate 2-octanone was 50 mM. The substrate solution was prepared by dissolving in phosphate buffered solution  and a glutamic acid-rich leucine zipper motif (ZE)  . The devR-ω-Transaminase (RTA) and alanine dehydrogenase (AlaDH) are dimeric enzymes, each consisting of two subunits. ZE was fused to the C-terminal domain of the split NpuDnaE intein (IntC), the ZE-IntC segment was fused to the N-terminus of the RTA subunit (RTAS), constructing ZE-IntC-RTAS. ZR was fused to the N-terminal domain of the split NpuDnaE intein (IntN), the ZR-IntN segment was fused to the C-terminus of the AlaDH subunit (AlaDHS), constructing ZR-IntN-AlaDHS  are required, in order to reduce the steric hindrance for the subunit splicing. Dimerization of ZE and ZR can naturally occur due to the coiled-coil association between ZE and ZR  with the AlaDH subunit (AlaDHS), the ligated subunit of RTAS-AlaDHS. The molecular weight of RTAS-AlaDHS is 75.9 kDa. The band of RTAS-AlaDHS is below the position 97.2 kDa of a mark protein. The band indicated by the black arrow in S with the FDH subunit (FDHS). The band of the ligated subunit AlaDHS-FDHS has a molecular weight of 83.1 kD. Appearance of the bands of RTAS-AlaDHS and AlaDHS-FDHS confirmed the ligations of RTAS with AlaDHS and AlaDHS with FDHS, through the in vivo posttranslational hetrosubunit splicing triggered by the dimerization of ZE and ZR.The splicing products RTA&AlaDH and AlaDH&FDH were purified sequentially by Ni-Sepharose beads, ultrafiltration fractionation, and gel filtration chromatography. They were analyzed through SDS-PAGE . The banN-RTAS, and ZE-IntC-AlaDHS, the TMW of the possible ligation mode were predicated. We have tried to use matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to identify the fractions of the splicing products RTA&AlaDH and AlaDH&FDH. However, the measured data are not useful, due to the lack of reliability of the data. This is ascribed to the large molecular weights of the fractions of RTA&AlaDH and AlaDH&FDH. In an alternative approach, blue native polyacrylamide gel electrophoresis (BN-PAGE) was used to analyze RTA&AlaDH and AlaDH&FDH. BN-PAGE has been proved to be effective for identifying proteins from multiprotein complexes . The BN-S-AlaDHS  exchange was the external perturbation. The main correlation peaks observed in the synchronous and asynchronous maps of –1) exhibited a faster H-D exchange than the α-helix (1650 cm–1) and β-sheet (1610 cm–1) structures. For RTA#AlaDH, the cross-peaks  and  can be identified from the synchronous and asynchronous maps are shown –1) exhibited a faster H-D exchange than the α-helix (1639 cm–1), and the unordered structure represented by 1437 cm–1 exchanged before the β-sheet structure (1665 cm–1). For RTA + AlaDH (–1) exhibited a faster H-D exchange than the α-helix (1645 cm–1), and the unordered structure represented by 1427 cm–1 exchanged before the β-sheet structure (1680 cm–1).Infrared spectra as a function of the deuteration time were measured as shown in  + AlaDH , the cro2O. That is the unordered structures exhibited a faster H-D exchange than the structures of α- helices and β-sheets. Ascribing to the presence of strong hydrogen bonds in α-helices and β-sheets, the protons of α-helix and β-sheet are more difficult to exchange than those of unordered structures . Kd value of 0.68 ± 0.15 mM, which is smaller than the Kd value of 2.33 ± 3.22 mM by RTA#AlaDH, and much smaller than the Kd value of 14.91 ± 10.13 mM by RTA+AlaDH. It is indicated that RTA&AlaDH exhibited the affinity to 2-octanone 3.4 times stronger than that by RTA#AlaDH, and 6.5 times stronger than that by RTA+AlaDH.The analysis of enzyme binding to substrate can provide the information of enzyme affinity toward the substrate. Microscale thermophoresis (MST) is a relatively recent technique based on the unique physical principle of thermophoresis. It can be used to quantify the interactions of proteins and small molecules . The twoIn the conversion of the ketones to amines under the catalysis of RTA, alanine was used as amine donor, and pyruvate being the co-product. Maintaining alanine with a low concentration facilitates subsequent purification of the product. In addition, recycling of pyruvate back to alanine can reduce the inhibitory effect of by-product. Alanine with a small concentration (5 mM) was used, which is tenth of that of 2-octanone (50 mM). The conversion was catalyzed under the three-enzyme systems RTA&AlaDH + AlaDH&FDH, RTA#AlaDH + AlaDH#FDH, and RTA + AlaDH + FDH. + between AlaDH and FDH cannot be directional. In contrast, RTA&AlaDH + AlaDH&FDH and RTA#AlaDH + AlaDH#FDH achieved the paired hetrosubunits, RTAS-AlaDHS and AlaDHS-FDHS, as illustrated in + and recycling alanine and NADH. Thus the transferring and recycling are directional and oriented. The hetrosubunit pairing through peptide bonds makes the splicing products become stable in comparison to the mixed enzymes. In addition, the splicing products have exhibited much higher affinity toward the model substrate 2-octanone. All these contribute to the splicing products exhibiting much higher catalysis efficiency than the mixed enzymes.For the mixed enzymes RTA + AlaDH + FDH, the interactions between the enzymes are not specific interactions. Under shaking condition during the reaction, occasionally pairing of RTA with AlaDH and AlaDH with FDH occurred. However, the instantaneous pairing of the enzymes cannot be controlled and maintained for the whole reaction time, attributed to the non-specific interaction between the enzymes. This results in that, the transfers of alanine and pyruvate between RTA and AlaDH and NADH and NADS-AlaDHS in RTA&AlaDH is 28.5% higher than that in RTA#AlaDH, and the number of AlaDHS-FDHS in AlaDH&FDH is comparable to that in AlaDH#FDH. Possibly, this is the reason that RTA&AlaDH contains more alpha-helical structure than RTA#AlaDH. It is implied that, RTA&AlaDH has more alanine→pyruvate→alanine cycles than RTA#AlaDH. Due to having more pairing hetrosubunits, RTA&AlaDH + AlaDH&FDH is more stable than RTA#AlaDH + AlaDH#FDH. In addition, RTA&AlaDH + AlaDH&FDH exhibited much higher affinity toward the substrate 2-octanone than RTA#AlaDH + Ala DH#FDH. As a result, RTA&AlaDH + AlaDH&FDH exhibited a higher catalysis efficiency than RTA#AlaDH + AlaDH#FDH.Based on the BN-PAGE analysis for RTA&AlaDH, RTA#AlaDH, AlaDH&FDH, and AlaDH#FDH, the linking mode corresponding to each band has been determined, and the fraction of each band has also been obtained. Based on the information, the number of the pairing hetersubunits RTAin vivo hetrosubunit splicing, and the pairing enzymes RTA&AlaDH and AlaDH&FDH have been constructed. The hetrosubunit splicing has little effect on the intro-hydrogen bonding interactions, and can confer RTA&AlaDH having more alpha-helical structure and a higher stability. Ascribing to the hetrosubunit pairing through peptide bonds, the transferring pyruvate and NAD+ and recycling alanine and NADH are directional and oriented. In contrast to the mixed three enzymes RTA+AlaDH+FDH, the ligated enzymes RTA&AlaDH+AlaDH&FDH exhibited a much larger substrate affinity, higher stability, and significantly enhanced activity.The heterodimerization of leucine zipper motifs promoted the All datasets generated for this study are included in the article/E-IntC-link-AlaDH/RTA-link-IntN-ZR-pETDuet and ZE-IntC-link-AlaDH/FDH-link-IntN-ZR-pETDuet, performed the experimental setup and procedure. RL, YC, and KD carried out the analysis and evaluation of the results. All the authors approved the final manuscript.RL and WF designed the experiment study and prepared the manuscript. RL contributed the expression plasmids ZThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The classic ketogenic diet (cKD) requires constant nutritional monitoring over time both to ensure its effectiveness and to reduce the likelihood of short- and long-term adverse effects. We retrospectively described the use of remote monitoring by e-mail during the first year of follow-up on cKD in 34 children  diagnosed with drug-resistant epilepsy  or glucose transporter type 1 deficiency syndrome . All the e-mails were evaluated analyzing their frequency and content at 3, 6 and 12 months. Three families never sent e-mails. A median of 36.0 (IQR 23.0–64.0) e-mails per family were sent during the 12 follow-up months by the 31 patients. GLUT1-DS patients sent a greater number of e-mails than the DRE group (median 39.0 (IQR 25.5–56.5) vs. median 26.0 (IQR 19.0–65.0)). At the end of the follow-up period, a greater number of e-mails had been exchanged between the nutritional team and the families belonging to the group that increased its linear growth , compared to the other ones. Constant remote monitoring by e-mail could be a feasible and effective way for a better cKD management. Ketogenic dietary therapies (KDTs) are well-established, non-pharmacologic treatments used for children and adults with medication-refractory or drug-resistant epilepsy (DRE) , but theThe recommendations of the International Ketogenic Diet Study Group about optimal clinical management reported that a multidisciplinary team—including pediatric neurologists, nutritionists, dietitians and pediatricians—should closely monitor patients to maximize the outcome of the therapy . Phone oBased on our experience, careful long-term monitoring of dietary and clinical parameters is essential to support compliance, investigate possible side effects and promptly deal with them. There is a need for a solution that can facilitate more convenient and easy communication between families and physicians for a better therapy management and ensure proper patient follow-up, regardless of the distance.To date, many experiments have confirmed the remarkable achievements of telehealth ,10,11, wRecent studies have demonstrated the feasibility and efficacy of telemedicine in the field of neurology, also referred to as “teleneurology”, including in the care of patients with epilepsy ,14,15.Teleneurology is the use of modern communication technology to enable treatment when the doctor and patient are not present in the same place, and potentially not at the same time. The two main communication technologies used are videoconferencing equipment and e-mails, where the consultation is carried out without the patient being present and at a time convenient to the doctors involved . The eviTo our knowledge, the actual use of remote monitoring by e-mail has never been investigated before in patients on classic ketogenic diet treatment. The aim of this study is to retrospectively describe the use of remote monitoring by e-mail during the first year of follow up on cKD in 34 patients with GLUT1-DS and DRE. We wanted to evaluate the number and content of e-mails firstly in the whole group of patients and secondly to compare the results of subgroups of patients divided by diagnosis and growth pattern after 1 year on the diet.The study protocol complied with the tenets of the Helsinki Declaration and was approved by the ethical committee of the Fondazione IRCCS Policlinico San Matteo of Pavia on 13 May 2019 (code number: 20190033749). All patients and/or caregivers provided their written informed consent.All participants were consecutively enrolled at the Department of Child Neuropsychiatry of the IRCCS Casimiro Mondino Foundation  between 2008 and 2017. Inclusion criteria were as follows: (1) age between 2 and 17; (2) diagnosis of DRE (defined as unresponsiveness to at least two anticonvulsant medications) or GLUT1-DS; and (3) treatment with cKD for at least 12 months. Children with severe organ failure, thyroid disorders, or requiring enteral and parenteral nutrition were excluded. Clinical data were extracted from a retrospective review of clinical records and entered onto an electronic database.Using a standardized approach , the HumOral citrates were also administered to prevent the formation of kidney stones. All patients started at home on a 1:1 ketogenic diet, with ketogenic ratios subsequently increased to 2:1, 3:1, or 4:1 in order to achieve blood β-OHB levels ≥ 2.0 mmol/L. Family members were instructed to monitor blood β-OHB concentrations (twice per day every day during the induction phase and then once or twice a week) and report values by e-mail to the study investigators.All participants underwent control visits 1 month after cKD initiation, then after 3, 6, 9, and 12 months, respectively. At follow-up visits, each patient underwent (1) measurements of fasting blood ketones, (2) assessments of compliance to the prescribed dietary regimen and supplement use, and (3) screening for potential adverse effects.After the beginning of the diet and during the time intervals between control visits, parents or caregivers were asked to send e-mails in order to evaluate patients’ clinical response to the therapy and adjust ketogenic ratio prescription if needed. They were asked to report on diet compliance, beta-hydroxybutirate levels, side effects, dietary change requests or doubts about cKD management and supplement use.All the e-mails exchanged between the patient’s family and the keto-team during the follow-up period after the beginning of the diet were analyzed. The total number, median frequency and e-mail contents were examined and collected in an Excel database. In particular, the total e-mail number and specifically those regarding dietary change requests (diet/menus modifications), ketonemia monitoring, adverse effects, supplements or drugs (information about the sugar content in supplements or changes with other products proposed by caregivers), blood chemistry, dietary info requests  and cKD suspension were analyzed and divided in 3-month periods , for the entire year of treatment.The use of e-mail monitoring was analyzed in the group as a whole and in subgroups created according to diagnosis and growth pattern after one year on the diet .Quantitative variables were summarized by medians and interquartile ranges (IQRs), qualitative variables were summarized through percentages. The comparison across the three growth groups was performed using the non-parametric Kruskal–Wallis test, while to compare characteristics in different disease groups the Mann Whitney test was carried out. Associations between categorical variables were analyzed with the Chi-squared test or the Fisher’s exact test or the Fisher–Freeman–Halton’s exact test, as appropriate. The software used for the analysis was STATA/SE® for Windows, version 15 . Two-tailed p values < 0.05 were considered statistically significant.p = 0.041) and began dietary treatment earlier after diagnosis  years, vs. median 4.7 (IQR 3.5–6.5) years, respectively, p < 0.001)—an expected finding, since cKD is the only available treatment for GLUT1-DS.Patients with GLUT1-DS were diagnosed at an older age (median 8.5 (IQR 5.0–10.0) years) than those with DRE (median 4.0 (IQR 1.0–7.5) years; There were significant differences in terms of anti-epileptic drugs (AEDs) and neurological impairment between the two groups. Specifically, patients with GLUT1-DS had minimal neurological impairment and none of them were taking AEDs. Ten patients with DRE were on multiple anti-epileptic drugs (AEDs) and their pharmacological treatment was not modified throughout the study. Most of the patients with DRE had severe neurological impairment.The linear growth of the patients included in the present study has been described in a recent paper . AccordiThree families never sent e-mails but maintained only phone contact, so they were excluded. The sample reduced to 31 patients.As regards the whole sample , a mediaThe content of the e-mails concerned primarily general or specific diet-related questions, a median of 16.0 e-mails (IQR 6.0–31.0) for patient were collected; families often had doubts about food choices  or how to manage hunger .A median of 6.0 e-mails related to both weekly or monthly registered ketonemia levels (IQR 4.0–17.0) and diet/menus modifications (IQR 2.0–13.0). We investigated possible correlation between blood β-OHB levels and total number of sent e-mails, but no significant differences emerged (data not shown).A very low number of e-mails (median 1.0 (IQR 0.0–3.0)) concerned adverse effects  or supplements (like information about the sugar content of supplements or changes with other products proposed by caregivers).No cKD suspension requests were sent during the entire year of treatment, except for one family.E-mail response time by the nutritional team depended on the kind of request. “Simple” questions or topics  took a relatively brief time lapse, so we managed to give an answer within the same day. For more complex topics  that required team collaboration and decision-making, the time lapse could be more extended (from 2 days to one week).The study sample was composed of 13 subjects (42%) affected by DRE and 18 patients (58%) diagnosed with GLUT1-DS.The use of e-mail monitoring was compared between these two groups of patients  and it wThe amount of e-mails relating to dietary information differed between the two groups, although not in a statistically significant way (median DRE 8.0 (IQR 3.0–31.0) vs. GLUT1-DS median 18.0 (IQR 11.0–29.5)), while no request for diet suspension was e-mailed by either group.The e-mails relating to adverse effects were greater in the DRE group after 12 months compared to the GLUT1-DS (median 3.0 (IQR 0.0–4.0) vs. median 0.0 (IQR 0.0–2.5)), although no significant statistical difference was reported.The use of e-mails was compared among the three subgroups based on the growth pattern .At the end of the follow-up period, a greater number of e-mails had been exchanged between the nutritional team and the families belonging to Group I (median 83.5 (IQR 48.0–102.0)) compared to Groups D and T (median 40.0 (IQR 21.0–65.0) and 30.0 (IQR 21.0–49.0), respectively), although no significant statistical difference was found.This trend was found in all e-mail subgroups, but especially in the number of e-mails requiring information about diet management: a median of 28.5 e-mails (IQR 18.0–49.5) in Group I, 18.0 e-mails (IQR 8.0–34.0) in Group D and 11.0 e-mails (IQR 3.5–26.5) in Group T.All groups sent a substantial number of e-mails about dietary change requests (menu modification) with a median of 19.5 e-mails (IQR 11.5–24.5) in Group I, 3.0 e-mails (IQR 0.0–6.0) and 5.5 e-mails (IQR 2.5–10.5) in Group D and Group T, respectively.The e-mails relating to the sending of ketonemia values were greater in Group I, although no significant statistical difference was found. In particular, a median of 19.0 e-mails (IQR 9.0–33.5) was found in Group I, 6.0 e-mails (IQR 2.5–9.0), 6.0 e-mails (IQR 3.0–26.0) in Group D and Group T, respectively.In this study, we retrospectively analyzed the use of remote cKDs monitoring by e-mail during the first year of follow-up in 34 patients with GLUT1-DS and DRE.Three families did not keep e-mail contact during the study period, instead they maintained only phone contact, so they were excluded and the final sample was composed of 31 families.All the exchanged e-mails between the patient’s family and the nutritional team were examined, both in terms of frequency and content, at 3, 6 and 12 months after cKD initiation.Our data showed that, in each period considered, GLUT1-DS families sent a greater number of e-mails compared to the DRE group. Specifically, in the first three months of diet, the GLUT1-DS group asked for diet modifications and dietary information more frequently than DRE patients. This trend could be explained by the fact that KD is actually the only therapy currently available for GLUT1-DS patients, who may be more motivated to a strict dietary adherence. Another explanation could be that GLUT1-DS patients had a minimal neurological impairment and a higher median age, so they could be more involved in choosing their menu. During the follow-up period, DRE patients’ caregivers sent a greater number of e-mails referring to adverse effects such as nausea, general tiredness, and vomiting. For those patients, the ketogenic diet is actually an alternative therapy, so it is possible that families tend to emphasize the onset or persistence of side effects. Another important factor to take into account is that DRE patients had severe neurological and clinical impairment that could have negatively influenced diet tolerability.During the first 3 months, phone or e-mail contacts between patient/caregiver and the team are surely more frequent, since this is the induction period, during which greater support is needed in order to try to solve—in the shortest time possible—any difficulties or doubts regarding diet management. Moreover, during the first period, ketonemia levels and clinical response to the treatment are often inconstant and require a stricter contact with the family.From the nutritional point of view, the most relevant topics expressed in the e-mails were dietary changes and info requests, ketonemia monitoring, reported adverse effects and multivitamin–mineral supplements information.Lack of palatability of ketogenic food, as well as qualitative and quantitative restrictions in meal preparation, can be burdensome. Each ingredient needs to be weighed per gram, resulting in a long time spent for meal preparation; the overall treatment should be continuously monitored and tailored to the individual patient. A great aid to families is given by applications available for smartphones or software for personal computers, through which an appropriate count of nutrients can be easily calculated for each meal and recipes approved by dieticians can be shared. Adherence to cKD implies important limitations in participation in daily social activities, not only for patients but also for their families . DespiteIn our previous study, we speculated that our promising results on growth may depend on an accurate assessment of energy needs , an adequate protein-to-energy ratio according to WHO recommendations, as well as the continuing support offered to caregivers involved in the management of KD . In the On this basis, we can hypothesize that constant monitoring during the first year of ketogenic diet could result in a greater adherence to the treatment.The hypothesis that e-mail monitoring could be effective in ketogenic diet management was investigated only in one previous study conducted by Cervenka C.M. and colleagues a few years ago . AlthougAs reported in recommendations of the International Ketogenic Diet Study Group about optimal clinical management, phone or e-mail contact within the first month is advised . ConstanE-mail monitoring has a number of distinct advantages; for example, it can be read and accessed from different sites, so that the dietician or the physician can be in contact with the patient both from the hospital and from home/wherever he/she is and rapidly answer patients’ requests, if necessary. Using a structured pro forma to record the history and the basic neurological examination, it is possible to triage referrals into those patients who can be reassured, those who require straightforward investigations and those who need to be seen. In addition, this system enables a much better two-way communication between hospital specialists and other colleagues .Another advantage could be that, in this way, the patient is monitored and rapidly helped by the nutritional team. In fact, e-mail contact allows for an easier communication with the dietician  and to make any changes to the menus based on patients’ tastes and/or dietary prescription, increasing cKD adherence.The caveats of our investigation include its retrospective design, the limited sample size and follow-up period, and the inability to detect telephone contacts that integrate monitoring. Moreover, the lack of a control group did not permit an investigation of the effects on compliance or efficacy.These limitations notwithstanding, our study describes for the first time the use of remote monitoring during ketogenic diet treatment in DRE and GLUT1-DS patients.Constant monitoring via email use by families, especially during the first months of the ketogenic diet, could in our experience actually lead to greater compliance by the patient and consequently have a positive effect on the normal growth process. Further studies are needed on the use of this type of remote follow-up, taking into account the cost/benefit ratio of the inclusion in the routine therapeutic process. Moreover, it would be useful and desirable to conduct future prospective case–control studies to evaluate the effect of monitoring via e-mail on adherence to the ketogenic diet.Constant remote monitoring by e-mail could be a feasible and effective way for a better KDTs management, especially in those patients who live far from the reference care center. It seems to be very useful in the first 12 months of therapy, to support and help families facing for the first time all the aspects of cKD treatment.


Haemaphysalis longicornis (H. longicornis). MicroRNAs (miRNAs) participate in the development of organisms by inhibiting the expression of their target mRNAs. In this study, a novel tick-specific miRNA was identified and denoted hlo-miR-2 that serves as a novel regulator of molting events in H. longicornis nymphs by targeting a cuticular protein. The full length of this cuticular protein was first obtained and named it CPR1. A qRT-PCR analysis showed that hlo-miR-2 and CPR1 exhibit significant tissue and temporal specificity and that their transcription levels are negatively correlated during the molting process. CPR1, as a direct target of hlo-miR-2, was identified by a luciferase reporter assay in vitro. Agomir treatment indicated that the overexpression of hlo-miR-2 significantly reduced the protein expression level of CPR1, decreased the molting rate and delayed the molting time point in H. longicornis nymphs. RNA interference (RNAi) experiments demonstrated that CPR1 was significantly associated with the molting process in H. longicornis nymphs. Phenotypic rescue experiments convincingly showed that hlo-miR-2 participated in molting events by targeting CPR1 in H. longicornis nymphs. In summary, we present evidence demonstrating that miRNAs constitute a novel important regulator of molting events in addition to hormones. The described functional evidence implicating CPR1 in molting events contributes to an improved understanding of the distinct functions of the CPR family in ticks and will aid the development of a promising application of cuticular protein RNAi in tick control.Successful completion of the molting process requires new epidermal growth and ecdysis of the old cuticle in  H. longicornis requires three hosts and undergoes two molting processes during its lifetime . A histological study of the tick cuticle ultrastructure has shown that the tick cuticle consists of two parts, the epicuticle and the procuticle, and the procuticle consists of two layers, namely, the inner and the outer endocuticle  are non-coding RNAs with a length of approximately 22 nt that posttranscriptionally regulate their target genes by binding to the 3′ untranslated regions (UTRs) of the corresponding mRNAs . Since tgicornis .H. longicornis at different developmental stages. Among these miRNAs, a potential novel miRNA, named novel miRNA-2, exhibited high expression at the unengorged larval, unengorged nymph and unengorged adult developmental stages but not at the egg stage. Based on this finding, we speculated that novel miRNA-2 might play an important role in the maintenance of the developmental state of H. longicornis. Therefore, this study aimed to reveal the biological role of novel miRNA-2 in the development of H. longicornis.In our previous study, various miRNAs, including conserved and potentially novel miRNAs, were identified by deep sequencing of H. longicornis were established by the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China (LVRI). The ticks were reared at 25°C in cages with 90% relative humidity and allowed to feed on the blood of New Zealand white rabbits. Under a stereoscopic microscope , semi-saturated H. longicornis was dissected, and the tissues in the body cavity  were isolated and collected. Epidermal tissue was obtained by flushing with RNase-free water to remove the contents of the residual body cavity. A study conducted by H. longicornis nymphs lasts 16.9 days, whereas our study revealed a duration of 15.47 days (Colonies of .47 days . TherefoMT274605) were produced using a PrimeScriptTM RT Reagent Kit (Takara) according to the manufacturer’s recommended protocol. All primer designs were performed using Primer Premier v5.0, and the primer details are shown in ®Premix EX TaqTM II Kit  according to the manufacturer’s instructions. Each sample was measured in triplicate, and the relative expression levels were calculated using the 2–ΔΔCt method and normalized to the expression of the housekeeping gene β-actin.Total RNA was extracted using the TRIzol reagent . cDNAs of the mature sequence of novel miRNA-2 and the CP CPR1 . For partition function folding, the algorithm disallows pairs that can only occur in isolation, and other pairs might still occasionally occur as helices of length 1  technique was used to amplify the full length of the target gene. The two pairs of GSP primers were used to amplify the 3′ UTR and 5′ UTR of the CPR1 according to the RACE kit instructions . All the primers were designed using Primer Premier v5.0, and the primer details are shown in Three miRNA target prediction programs, namely, RNAhybrid® Luciferase Assay System (Promega) according to the manufacturer’s instructions. The firefly luciferase activity of the pmirGLO vector was measured for the normalization of Renilla luciferase activity. The treatments were performed in triplicate, and the transfections were repeated three times (293-T cells (cultured by LVRI) were grown in DMEM  supplemented with 10% fetal bovine serum (FBS)  and 1% 1x antibiotic-antimycotic (Gibco). Luciferase constructs were generated by inserting the 3′ UTR of the CPR1 gene into the pmirGLO vector . A pair of primers was used to amplify the 3′ UTR of CPR1 and were designed using Primer Premier v5.0. The primer details are shown in ee times .For the assessment of novel miRNA-2 and CPR1 colocalization, antisense RNA detection probes targeting novel miRNA-2 and the CPR1 gene were designed and labeled with the dual fluorophores Cy3 and FAM, respectively, at Servicebio Biotechnology Company . A scrambled sequence and the sense probe for the target gene were used as NCs. Two days after microinjection, the tick nymphs were dissected in cold PBS buffer, fixed overnight in 4% paraformaldehyde, and incubated overnight at 37°C for probe hybridization . The ticTM agomir negative control #22 (NC), antagomir (Ant), and micrOFFTM Ant negative control #22 (NC) were synthesized by RiboBio Company .Agomir (Ag), micrONH. longicornis nymphs were dosed at a site between the third and fourth legs. The experimental group was administered 800 μM novel miRNA-2 Ag or Ant in 0.2 μL of RNase-free water, the NC group was administered 800 μM micrONTM Ag or micrOFFTM Ant in 0.2 μL of RNase-free water, and no injection was administered to the blank control group. Each group included 50 H. longicornis nymphs collected 5 days after blood meal ingestion.Five days after blood meal ingestion, TM reagent  in accordance with the manufacturer’s instructions. Moreover, the number of successfully completed molts and the molting timepoints after microinjection were observed and recorded.Thirty ticks were used for observation of the molting timepoint and molting rate, and the rest were used to detect the expression of related genes and proteins. Two days after injection, total RNA and protein were extracted from the ticks using the TRIzolH. longicornis sequence, was synthesized by RiboBio . Five days after blood meal ingestion, H. longicornis nymphs were dosed at a site between the third and fourth legs: the experimental group was administered 800 μM iCut in 0.2 μL of RNase-free water, and NC group was administered 800 μM NC iCut in 0.2 μL of RNase-free water, and no injection was administered to the blank control group.The double-stranded siRNA targeting CPR1 (iCut) and the NC for iCut, which exhibited no significant sequence similarity to the In the rescue experiments, the tick nymphs were divided into four groups and treated as follows: coinjection with 0.2 μL of an Ant/iCut mixture (800 μM Ant and 800 μM iCut), coinjection with 0.2 μL of an Ant/NC iCut mixture (800 μM Ant and 800 μM NC iCut), coinjection with 0.2 μL of an NC Ant/iCut mixture (800 μM NC Ant and 800 μM iCut), and coinjection with 0.2 μL of an NC Ant/NC iCut mixture (800 μM NC Ant and 800 μM NC iCut).H. longicornis nymphs obtained 5 days after blood meal ingestion. Thirty ticks were used for observation of the molting timepoint and molting rate, and the rest were used for the detection of related genes and proteins. Two days after injection, total RNA and protein from the ticks were extracted using the TRIzolTM reagent  in accordance with the manufacturer’s instructions. Moreover, the number of successfully completed molts and the molting timepoints after microinjection were observed and recorded.Each group included 50 TM reagent  according to the manufacturer’s instructions, separated on 12% SDS–PAGE gels and transferred to PVDF membranes . The membranes were then blocked in TBST containing 5% skim milk for 1 h at room temperature. For the detection of CPR1, the membranes were incubated overnight at 4°C with monoclonal antibodies against CPR1 at a 1:300 dilution and then incubated with a 1:5000 dilution of an anti-rabbit HRP-conjugated secondary antibody  at room temperature for 1 h. CPR1 expression was detected by chemiluminescence .Total protein from the ticks was extracted using the TRIzolThe samples were fixed in 3% glutaraldehyde at room temperature for 2 h and transferred to 4°C for storage. The samples were fixed in 1% osmium tetroxide for 2 h, dehydrated in an alcohol gradient , and incubated with isoamyl acetate for 15 min. The samples were then dried in a K850 critical point dryer  and subjected to gold sputtering for approximately 30 s with an MSP-2S instrument . Observations and imaging were performed under an SU8100 scanning electron microscope  .t-test or the Wilcoxon test based on the following significance levels: ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ****P < 0.0001.All the data were analyzed using GraphPad Prism 7, and the results are presented as the means ± SEMs. The F test was used for equal variance detection, and the means were compared using Student’s The potential novel miRNA-2 needs to be further characterized and validated. One of the most notable characteristics defining a miRNA gene is a precursor with a typical stem-loop structure, and at most four symmetrical mismatches within the duplex and at most two asymmetric bulges of up to 2 bases are allowed . Close sH. longicornis. The results demonstrated that novel miRNA-2 was abundantly expressed in both the midgut and epidermis but was expressed at low levels in the other tissues   .Three publicly available bioinformatic tools for miRNA target gene prediction were used to predict the target gene of novel miRNA-2. The same prediction results obtained from all of the tools showed that the CPR1 gene contains a binding site for novel miRNA-2. We obtained the full-length sequence of the CPR1 gene using RACE technology. The full length of the CPR1 gene contained 1171 bp, and the coding sequence (CDS) was 339 bp in length (Supplementary Data 1). The putative novel miRNA-2-binding site in CPR1 is located at position 661, which is located at the 3′ UTR of CPR1, and this binding is a classical binding mode .in vitro , and as expected, the expression in the NC and blank control groups was unaffected. Moreover, the Western blot results showed that the expression of CPR1 in the Ag-treated group was lower than that in the NC (0.989 ± 0.05615 fold) and blank control (0.979 ± 0.06109 fold) groups, which suggested that overexpression of novel miRNA-2 can downregulate CPR1 expression.To determine the function of novel miRNA-2 in molting events, we overexpressed novel miRNA-2 by microinjecting Ag into t-test, P < 0.0001). Moreover, the molting rate of the Ag-treated group was markedly decreased to 73.33% of the value obtained for the NC and blank control groups. These results strongly indicated that novel miRNA-2 overexpression exerts a negative effect on the molting process in H. longicornis nymphs   compared with the other groups, and no significant difference was observed between the NC (0.985 ± 0.05381) and blank control (0.973 ± 0.0771) groups. However, the Western blot analysis showed that the protein expression level of CPR1 was increased by Ant treatment compared with that found in the NC and blank control groups, which suggested that the blockage of novel miRNA-2 expression can upregulate CPR1 expression.To decipher the role of novel miRNA-2 in molting events, we blocked the expression of novel miRNA-2 by microinjecting Ant into t-test, P < 0.0001). Moreover, the molting rate did not differ significantly between the Ant-treated and NC control groups (The molting timepoint of the Ant-treated group was reduced to 14.39 ± 0.1654 days compared with that of the NC group (15.4 ± 0.1633 days)  compared with other group and not appreciably differ between the NC (0.996 ± 0.03407) and blank control (0.984 ± 0.02575) groups, Moreover, the Western blot analysis showed that the protein expression level of CPR1 was decreased by iCut treatment compared with that found in the NC and blank control groups, which suggested that a siRNA designed to target CPR1 successfully decreased the CPR1 transcript and protein levels in H. longicornis nymphs.Because CPR1 was expressed in the epidermis and its expression changed during the normal molting process of t-test, P < 0.0001), whereas no significant difference in the molting timepoint was observed between the NC and blank control groups  , but this delay was shorter than that obtained for the iCut/NC Ant group (21.4 ± 0.3625 days) . However, the molting rate of the Ant/iCut group was notably declined to 76% of that obtained for the Ant group but was higher than that of the iCut group (46.67%)  (Student’s (46.67%) . These rH. longicornis. The full-length novel miRNA-2 precursor has a length of 62 nt and exhibits a typical stem-loop structure, and its mature sequence is 22 nt in length. We named this miRNA hlo-miR-2.In this study, we first discovered and verified the presence of novel miRNA-2 in H. longicornis. Specifically, it is highly abundant in unengorged larvae, unengorged nymphs and unengorged adults but not in eggs. This miRNA is also highly expressed in epidermal and midgut tissues compared with other tissues.hlo-miR-2 exhibits substantial stage and tissue specificity in H. longicornis.Our results identified CPR1 as a target gene of hlo-miR-2 and showed that this gene was also highly abundant in epidermal tissue. Moreover, the qRT-PCR analysis showed that the levels of hlo-miR-2 and CPR1 were negatively correlated during the molting process of in situ hybridization results showed that CPR1 interacted directly with novel miRNA-2 in a spatially dependent manner.This study provides the first determination of the full-length sequence of CPR1, which was 1171 bp in length. The CDS was 339 bp in length, and the binding site for hlo-miR-2 was located in the 3′ UTR of CPR1. The miRNA/mRNA fluorescence The epidermis provides a barrier for the defense of ticks against adverse environmental changes and mechanical stimuli, and ticks must periodically shed and resynthesize their epidermis during development. Members of the CPR family, which is the largest family of CPs, are expressed in the procuticle and play a role in binding to chitin . A simulH. longicornis CPR1 3′ UTR with the hlo-miR-2 mimic resulted in decreased Renilla luciferase activity in vitro, which suggested that CPR1 is a direct target gene of hlo-miR-2 in vitro.As determined through dual luciferase reporter assay, the cotransfection of a luciferase reporter vector containing the H. longicornis nymphs, and markedly decreased the molting rate. The depletion of hlo-miR-2 in H. longicornis nymphs increased CP expression and accelerated the molting process in H. longicornis nymphs. These results suggested that hlo-miR-2 plays an important role in H. longicornis nymph molting events. The data obtained from the siRNA-mediated silencing of CPR1 during the H. longicornis nymph molting process suggested that CPR1 is necessary and sufficient for molting events.The overexpression of hlo-miR-2 decreased CPR1 expression, significantly delayed the molting timepoint of in vivo and counteract the inhibition of hlo-miR-2; thus, only the inhibitory effect of the CPR1 siRNA is reflected in the phenotype. Additionally, these results suggested that CPR1 significantly affects the formation of the original epidermis, whereas hlo-miR-2 has a significant inhibitory effect on the posttranslational level of CPR1, which indicated that CPR1 is a target gene of hlo-miR-2 in vivo.The phenotypic rescue experiment showed that the siRNA/Ant group exhibited a delayed molting timepoint and a decreased molting rate compared with the Ant group, which indicated that the Ant can bind to hlo-miR-2 H. longicornis 20-E titers increased sharply on day 6 after nymph engorgement, and this event was accompanied by the initiation of apolysis and the appearance of the exuvial space (Ornithodoros moubata and Amblyomma hebraeum (H. longicornis nymph engorgement, but this possibility needs further study.A histological study showed that the al space . Additiohebraeum . HoweverBombyx mori, Manduca sexta and locusts have suggested that the level of LmTwdl1, a member of the Tweedle family of CPs, gradually increased beginning on day 5 after nymph engorgement, which is a vital period for cuticle renewal, and that this gene has conserved essential functions in molting events (H. longicornis nymphs.Previous studies of g events . A studyH. longicornis that exhibits significant tissue and temporal specificity. The results from RNAi experiments showed that CPR1 is highly associated with the molting process in H. longicornis nymphs. More importantly, through dual luciferase reporter assays and phenotypic rescue experiments, we uncovered the role of hlo-miR-2 in molting events in H. longicornis nymphs. Furthermore, we identified CPR1 as a direct target of hlo-miR-2, and the obtained functional evidence verified that the hlo-miR-2-mediated regulation of CPR1 is essential for molting development in H. longicornis nymphs. In summary, our research indicates that miRNAs constitute a novel important regulator of the molting process in addition to hormones. The functional evidence implicating CPR1 in molting events contributes to a better understanding of the distinct functions of the CPR family in ticks and will aid the development of the promising application of cuticular RNAi in tick control.In conclusion, our study provides the first demonstration that hlo-miR-2 is an authentic miRNA in The datasets generated or analyzed during this study are included in this published article and its additional files.The animal study was reviewed and approved by Approval No. LVRIAEC2012-011.G-YL and W-GL designed the experiments. W-GL, Z-QQ, JL, Q-YR, Z-GW, JN, Y-CT, X-FQ, X-FX, H-LL, R-HX, and R-GC performed the experiments. W-GL analyzed the data and wrote the manuscript. G-YL, Z-QY, J-XL, SX, MR, HW, and HY critically revised the manuscript. All the authors read and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Osteomeles schwerinae C. K. Schneid.  is used traditionally for the treatment of various diseases in Asia. Previous studies have shown that OSSC elicits preventive effects in an in vivo model of diabetes. This study was to evaluate the antiapoptotic effects of dried leaves and twigs of OSSC extract and its major compounds in ARPE-19 cells—spontaneously arising human retinal pigment epithelial cells—under diabetic conditions. To examine the effects of an OSSC extract and its active compounds  on apoptosis in methylglyoxal -treated ARPE-19 cells and the mechanism by which these effects occur, apoptosis was measured using flow cytometry analysis. Protein expression levels of phospho-p53 (p-p53), Bax and Bcl-2 were determined by western blot analyses. The OSSC extract inhibited apoptosis in MG-treated ARPE-19 cells in a dose-dependent manner. The major compounds also reduced the rate of apoptosis. Both the extract and major compounds also inhibited the expression of p-p53 and Bax and increased the levels of Bcl-2 that had been previously reduced by MG treatment. The OSSC extract (0.1 μg/mL) and its major compounds (0.01 μM) attenuated apoptosis in ARPE-19 cells under toxic diabetic conditions by downregulating of expression of p-p53 and Bax. OSSC may serve as an alternative therapy to retard the development of diabetic retinopathy.The accumulation and formation of advanced glycation end products (AGEs) are related to diabetes and age-related disease.  The accumulation of advanced glycation end products (AGEs) is accelerated under conditions of chronic hyperglycemia and many age-related diseases . RetinalOsteomeles schwerinae C. K. Schneid. (OSSC), belonging to the family Rosaceae, is a perennial evergreen shrub, native to China that is harvested in the wild and used for local food  were further purified, as previously described [escribed . To checescribed .2 incubator, as previously described [The ARPE-19 cells were purchased from the American Type Culture Collection  and maintained at 37 °C in a humidified 5% COescribed .5 cells/well were treated with 1 mM MG in the presence or absence of OSSC extract and its major compound for 24 h. Following digestion with trypsin and washing twice with PBS, the cells were labeled with 5 μL Annexin V-FITC and 5 μL PI for 5 min each at room temperature for 15 min in the dark. After incubation, 400 mL binding buffer was added and the percentage of apoptotic cells was analyzed using a FACSCalibur flow cytometer .The rate of apoptosis was examined by flow cytometry using an annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) apoptosis detection kit  following the manufacturer’s protocol. ARPE-19 cells were placed into 6-well plates at the density of 2.5 × 10Polyacrylamide gel electrophoresis was performed, as described previously . MembranData are expressed as the mean ± S.E.M. ANOVA with Tukey’s test was used for multiple comparisons using Prism 5.04 software .In conclusion, we measured the antiapoptotic activities of the OSSC extract (0.1 μg/mL) and its three major compounds (0.01 μM) on MG-treated ARPE-19 cells, finding that the expression of the apoptotic factors p-p53, Bax and Bcl-2 was attenuated. Taken together, these results suggest that the use of the OSSC extract and its major compounds could prevent the development of DR.The patents related to this study were registered in Kore (No. 10-097394), China (No. XL200980160639.3), Hong Kong (No. HK1170958), England, France, Swiss, Germany (No. 247483), the United Arab Emirates (No. 1028) and the USA .


The psoralens 8-methoxypsoralen (8-MOP), 4,5′,8-trimethylpsoralen (TMP) and 5-methoxypsoralen (5-MOP) find clinical application in PUVA  therapy. PUVA treats skin diseases like psoriasis and atopic eczema. Psoralens target the DNA of cells. Upon photo-excitation psoralens bind to the DNA base thymine. This photo-binding was studied using steady-state UV/Vis and IR spectroscopy as well as nanosecond transient UV/Vis absorption. The experiments show that the photo-addition of 8-MOP and TMP involve the psoralen triplet state and a biradical intermediate. 5-MOP forms a structurally different photo-product. Its formation could not be traced by the present spectroscopic technique. The light-dependent PUVA  therapy is a well-established symptomatic treatment of skin diseases like psoriasis ,2, atopiThe mechanism of this photoaddition was addressed by steady-state  and timeHere, it will be investigated in how far the mechanistic picture derived from experiments on AMT can be transferred to psoralens  used in clinics. While for AMT a combination of time-resolved UV/Vis and time-resolved infrared (IR) spectroscopy was used to resolve the kinetics of the photo-addition, herein we use solely the technique of time-resolved UV/Vis spectroscopy available in our lab. The small quantum yields and/or water solubilities of these derivatives are very challenging for the small signals in time-resolved IR spectroscopy. It will be shown that for a comparison between derivatives the not as demanding and less resource-intensive method of time-resolved UV/Vis spectroscopy can still show distinct similarities and differences between derivatives. To this end, dissociation constants characterizing the intercalation, reaction quantum yields, spectroscopic patterns as well as kinetic parameters of the photo-addition were recorded. Based on the results, some guidelines for the rational improvement of PUVA agents shall be given. 20-3′ single strands. All three psoralen derivatives have, as opposed to AMT, low water solubility ranging from few micromolar for TMP to a few hundred micromolar for 8-MOP [The following results show spectroscopic measurements on psoralen derivatives with and without DNA. Synthetic DNA double strands with alternating adenine (A) and thymine (T) bases, AT-DNA in the following, were employed. They were formed by annealing 5′-(TA)or 8-MOP . The appor 8-MOP .D [D value represents a strong intercalation affinity. The dissociation constant KD can be determined by a titration experiment [D can be determined. The procedure relies on Equation (1) as well as Beer’s law and is specified in Reference [D value of 8-MOP and AT-DNA derived thereby amounts to 1.1 × 10−3 M. For 8-MOP and calf thymus DNA, Isaacs et al. have determined a similar value of 1.3 × 10−3 M [D values can be expected for differing DNA sequences and ionic strength of the sample [−4 M [D of 1.8 × 10−4 M for 5-MOP and AT-DNA was determined. A higher intercalation affinity compared to 8-MOP is in line with early reports [D determination by the above procedure difficult. Therefore, only the order of magnitude is estimated for the constant KD. For this estimate solid TMP was added to a solution of AT-DNA in amounts exceeding its solubility. Under these conditions the concentration D value of the order of 10−4 M was estimated.Intercalation of psoralens into DNA is a prerequisite for the photoaddition and, thus, has to be characterized. The propensity for intercalation is commonly quantified by the dissociation constant KD :(1)KD=cPperiment ,29 whichperiment . In the eference  for detaeference . The KD × 10−3 M . Deviatie sample . For AMTle [−4 M  was repo reports . The low−1 cm−1) and low or close to zero around 400 nm. The DNA has very low to zero absorption in this range. The spectrum of 8-MOP intercalated into AT-DNA shows an absorption up to 400 nm . The absorption coefficients are high at the lower end of the UVA . The diSimilar spectroscopic signatures are observed for the photoaddition of TMP to AT-DNA see b. Upon i of 0.12 . A highe of 0.12 ,31. In tThe spectral changes caused by the irradiation of intercalated 5-MOP are very different from the two discussed above c. The abA region . In compA region ,39. −1. It is attributed to the carbonyl stretching vibration [−1 and a more distinct one at 1591 cm−1 are attributed to C=C stretching vibrations. The spectrum of 5-MOP features a strong vibration band at 1733 cm−1 which can be attributed to the carbonyl stretching vibration [−1 can be assigned to C=C stretching vibrations. For the reason of IR transparency AT-DNA and 8-MOP were studied using buffer solutions based on D2O. This causes exchangeable protons of DNA to be replaced by deuterons, that is, NH vibrations are not to be expected. AT-DNA features four distinct bands in the upper-frequency range . The positive band at around 1750 cm−1 features a slight shift to higher wavenumbers with longer irradiation time  and AT-DNA (20 mM), 99% of 5-MOP is intercalated. At first sight, changes due to irradiation with a 375 nm LED f are sim−1). Acidic protons were exchanged for deuteron. After geometry-optimization, the wavenumbers and IR transitions strength were computed. The harmonic frequencies were scaled by a factor of 0.96 [Quantum chemical calculations support the interpretation of the experimental infrared spectra. Spectra were computed relying on density functional theory (DFT) using the B3LYP functional and a 6–31 + G* basis set as implemented in Gaussian 09 . The sel of 0.96 . −1 features the highest transitions strength  the double-stranded DNA by quantum chemistry were made. Instead only the IR spectrum of 1-methylthymine was computed. Three bands at 1661, 1624 and 1608 cm−1 can be seen. The ones with higher transition strengths are the carbonyl stretching vibrations, while the one at 1624 cm−1 can be assigned to a ring deformation mode [−1 experienced almost no shift. The carbonyl vibrations of thymine at 1667 and 1637 cm−1 are shifted by +6 and +29 cm−1 respectively. At 1591, 1584 and 1546 cm−1 the C=C stretching vibrations of 8-MOP can be seen, although the one at 1591 cm−1 has lost in strength as it is almost not visible. A synthetic difference spectrum was obtained by subtracting the computed 8-MOP and 1-methylthymine spectrum from the computed adduct spectrum. If one neglects the fact that the computed pattern is located at lower wavenumbers by ~−40 cm−1, the two patterns match rather well. The bleach at 1702 cm−1, although not as pronounced in the calculation, is due to the shifts in the carbonyl vibration of 8-MOP and one of the carbonyl vibrations of thymine. The positive feature around 1670 cm−1 in the experiment can be explained with the shift of the other thymine carbonyl vibration to higher wavenumbers. The bleach contributions at 1653 cm−1 and 1641 cm−1 seen in the experimental difference spectra are according to the computation due to carbonyl and C=C stretching vibrations of thymine. The fingerprint region is not very well matched, which at this level of calculation is plausible.For the calculation of the IR spectra of 8-MOP, Cambridge Structural Database (CSD) entry XANTOX was used as starting geometry . The carstrength a, green. compare a, thoughion mode . Base paion mode , explainion mode . Hereby,−1 and features the highest transition strength . The thymine vibrations are shifted by +10 and +33 cm−1 respectively. Bands at 1590 and 1570 cm−1 are attributed to C=C stretching vibrations of 5-MOP. The synthetic difference spectrum shows high similarity to the experimental one, with exception of the fingerprint region. The positive difference absorption around 1750 cm−1 is due to the shift of the 5-MOP carbonyl vibration, which cannot be seen in the calculation of the furan monoadducts. Hence, it can be seen as an indicator of the photoaddition on the pyrone side. For the calculation of the IR spectra of 5-MOP, CSD entry ARARIW was used as starting geometry . The carstrength e, green. compare b. The stSolutions of 8-MOP and TMP with AT-DNA were excited with UVA laser pulses and probed in the UV/Vis region. Due to the lack of spectroscopic signatures of the 5-MOP photo-product in the accessible UV/Vis region, only 8-MOP and TMP are treated in the following. 5 s-1), self-quenching (3.8 × 109 M−1 s−1) and quenching by oxygen (4 × 109 M−1 s−1) into account. For non-intercalated 8-MOP the signal at large delay times is essentially zero. For these delay times, the intercalated 8-MOP features a distinct difference absorption signal at wavelengths smaller than 360 nm.A solution of 8-MOP and AT-DNA in buffered water was excited with nanosecond laser pulses centered at 355 nm . At thesThe decay pattern of intercalated 8-MOP see , violet.ed 8-MOP , the timThe respective decay associated difference spectra (DADS) of the data above are shown in tion cf. , top.The photoreaction of TMP and AT-DNA was traced by the same approach. A solution of TMP and AT-DNA in buffered water was excited with nanosecond laser pulses centered at 355 nm . Due to d 470 nm . Indeed,Our previous results on the furan side photo-addition of AMT to AT-DNA  showed tFor 8-MOP intercalated into AT-DNA a bi-exponential decay with time constants of tion see  is connecess cf. . PresumaFor a triplet state as a precursor, the reaction quantum yield  be 0.06 . If thisFor the photo-product the signal t 370 nm  were use 0.3–0.4 ). Due toAlso, for TMP a bi-exponential decay pattern is observed. The time constants of o AT-DNA . Taking methanol . This wo for AMT .The behavior of 5-MOP is qualitatively different for the one of AMT, 8-MOP and TMP. In line with earlier studies, our UV/Vis and IR measurements indicate that the photo-addition proceeds via the pyrone side of the psoralen and not the furan side. Unfortunately, due to the lack of a spectroscopic signature of this adduct in the accessible UV/Vis range, the formation could not be traced in real-time. Time-resolved IR experiments ought to be conducted to clarify this in the future. 20-3′ was purchased from Sigma-Aldrich. The manufacturer purified the sample by HPLC. Annealing of the oligonucleotide strands in solution was performed within 24 h before the measurements by heating the solution in a water bath up to 93 °C and letting it cool down to room temperature within several hours. Solvents used were pure water , deuterium oxide  and acetonitrile-d3 . Solutions of the oligonucleotides were buffered with PBS . 8-MOP and 5-MOP were purchased from TCI  and TMP from Sigma-Aldrich . The lyophilized oligonucleotide 5′-(TA)2 windows of 3 mm thickness from Korth Kristalle were employed. The spectra were corrected for the absorption of aqueous vapor and HDO, if necessary. All measurements were performed at room temperature. Absorption spectra in the UV/Vis were recorded with a Lambda 19 spectrometer from Perkin Elmer. Fused silica cuvettes with path lengths of 0.1, 1 and 5 cm from Hellma were employed. Absorption spectra in the IR were recorded with an FT-IR-spectrometer Vertex 80v from Bruker Optik. A custom-made cuvette with a Teflon spacer for a path length of 0.1 mm was used. CaFPE were computed via the equation:For the irradiation in the steady-state experiments, LEDs emitting at 375 nm or 390 nm were used. For the determination of the quantum yield in the UV/Vis experiments the solutions were stirred while irradiating. With the light power Here, The nanosecond transient absorption data were acquired with a laser flash photolysis spectrometer LP980 from Edinburgh Instruments in a right-angle geometry. The frequency tripled output (355 nm) of a Nd:YAG laser  with a repetition rate of 5 Hz and a pulse duration of 12 ns (FWHM) was utilized for photoexcitation. The excitation energy ranged from 4–17 mJ per pulse. The diameter of the pump beam was ~8 mm. A pulsed xenon lamp (Osram XBO 150 W/CR OFR) generated the probe light. Fused silica flow-through cuvettes from Hellma with different path lengths in pump and probe direction were employed. To gain the best signal to noise ratio while keeping the turnover rate low, cuvettes with different dimensions  were used, depending on the sample. The transmitted probe light was dispersed by a grating monochromator and detected by a photomultiplier . The signal was digitized by an oscilloscope  and the absorption change was calculated based on measurements with and without laser excitation. For every time trace three sets of 8 consecutive measurements were averaged. If indicated, solutions were purged with oxygen or nitrogen . The measurements were performed at 17 °C. The time-resolved data were analyzed with a global multi-exponential fit functionDS) ΔAiλ .D [The photoaddition of three pharmaceutical psoralens  to AT-DNA was studied by steady-state and time-resolved spectroscopy. IR spectroscopy proved to be useful in distinguishing pyrone side (5-MOP) and furan side (8-MOP and TMP) photoadditions. The furan side additions were shown to proceed via a local triplet state and a triplet biradical. The present results, thus, underscore the importance of triplet states for the photo-reactivity. Once this state is populated, the investigated psoralens add to thymine with high efficiency. So, when optimizing psoralens for the PUVA therapy, a small dissociation constant KD , a low pD  and a hi


New therapies are urgently needed for Alzheimer’s disease (AD). Sodium oligomannate (GV-971) is a marine-derived oligosaccharide with a novel proposed mechanism of action. The first phase 3 clinical trial of GV-971 has been completed in China.We conducted a phase 3, double-blind, placebo-controlled trial in participants with mild-to-moderate AD to assess GV-971 efficacy and safety. Participants were randomized to placebo or GV-971 (900 mg) for 36 weeks. The primary outcome was the drug-placebo difference in change from baseline on the 12-item cognitive subscale of the Alzheimer’s Disease Assessment Scale (ADAS-cog12). Secondary endpoints were drug-placebo differences on the Clinician’s Interview-Based Impression of Change with caregiver input (CIBIC+), Alzheimer’s Disease Cooperative Study-Activities of Daily Living (ADCS-ADL) scale, and Neuropsychiatric Inventory (NPI). Safety and tolerability were monitored.p < 0.0001; effect size 0.531) after 36 weeks of treatment. Treatment-emergent adverse event incidence was comparable between active treatment and placebo . Two deaths determined to be unrelated to drug effects occurred in the GV-971 group.A total of 818 participants were randomized: 408 to GV-971 and 410 to placebo. A significant drug-placebo difference on the ADAS-Cog12 favoring GV-971 was present at each measurement time point, measurable at the week 4 visit and continuing throughout the trial. The difference between the groups in change from baseline was − 2.15 points (95% confidence interval, − 3.07 to − 1.23; GV-971 demonstrated significant efficacy in improving cognition with sustained improvement across all observation periods of a 36-week trial. GV-971 was safe and well-tolerated.NCT02293915. Registered on November 19, 2014ClinicalTrials.gov, The online version contains supplementary material available at 10.1186/s13195-021-00795-7. World Alzheimer Report 2018, approximately 50 million people worldwide are currently suffering from dementia, and two thirds of them have AD. China has more than 6 million AD patients, and this number is expected to exceed 10 million by 2050 -FDG-PET was carried out at two sites (Beijing and Shanghai) where appropriate technology and expertise were routinely available.This clinical trial was conducted at 34 participating sites in the psychiatry, neurology, and geriatric departments of hospitals in several regions of China. -FDG-PET sub-cohort underwent PET at baseline and week 36. We used the statistical region of interest (sROI) approach to compute the relative global CMRglu global index [Participants in the cerebral [al index . sROI waal index . The sROal index .p < 0.05 (two-sided) indicated significance.SAS v9.4  was used for statistical analyses. The screening, randomization, and follow-up of participants are summarized in Fig. The demographic and baseline characteristics of the trial cohort are summarized in Table p < 0.0001, with Cohen’s d effect size 0.53, using pooled SD of change score ANCOVA analysis. There were no significant drug-placebo differences for prespecified secondary outcomes. The p value of CIBIC+ was 0.059 between the groups    Figs. . The NPI0) Figs. .Table 2< 65; > 65 years) group, sex, education level , and MMSE score (3 terciles). In a post hoc subgroup analyses, significant intergroup differences were detected for CIBIC+ outcome in participants with MMSE scores 11–14 (p = 0.017), effect size 1.3.  participants in the GV-971 group and 31 (7.7%) participants in the placebo group were assessed by [In total, 307 of 407 participants (75.4%) in the GV-971 group and 303 of 410 (73.9%) in the placebo group had at least one treatment-emergent adverse event (TEAE) in the safety analysis set. Table Seventy-six participants (18.7%) in the GV-971 group and 86 (20.9%) in the placebo group reported a TEAE that was related or possibly related to the trial drug according to an investigator. Fourteen participants (3.4%) in the GV-971 group and 9 (2.2%) in the placebo group had a TEAE that led to their discontinuation from the trial.Thirty-three participants 8.1%) in the GV-971 group and 29 (7.1%) in the placebo group reported at least one serious adverse event (SAE). For the GV-971 group, the SAE of infectious pneumonia reported by one participant was determined as being possibly related to the trial drug by investigators. The remaining SAEs were determined to be not related or possibly related to the trial drug. Tables S% in the Two participants in the GV-971 group died during the trial because of metastatic lung cancer and brain stem encephalitis  of Drug Development Standards and Good Clinical Practice (GCP) guidelines, was organized and monitored by an international contract research organization (CRO), required centralized reading of MRI to confirm temporal lobe atrophy consistent with AD, and included extensive training in clinical trial methods by international vendors. GV-971 is an oligosaccharide with a novel proposed mechanism of action. In non-clinical studies, GV-971 showed a reduction of neuroinflammation in the brain by regulating the gut microbiota and reducing peripheral inflammation that may aggravate neuroinflammation. GV-971 directly binds to Aβ and decreases Aβ deposition in the brain –15. The In the full analysis set, none of the secondary endpoints, i.e., global function (CIBIC+), activities of daily living (ADCS-ADL) scale or behavioral symptoms (NPI), showed significant drug-placebo differences. The lack of significance on the CIBIC+, ADCS-ADL, and NPI might be attributable to the limited sample size, which was calculated based on the primary endpoint. Cultural differences may also have contributed to these negative outcomes as activities of daily living, and behavioral assessments are subject to many cultural interpretations . NPI scoA limitation of our trial was the lack of requirement for the presence of a diagnostic amyloid biomarker at screening, thereby potentially allowing participants who had dementia owing to non-amyloid-related diseases to be included. Amyloid positron emission tomography was not widely available in China at the time the trial was planned and initiated. Approximately 50% of the participants in the trial were carriers of the APOE4 gene and therefore had a higher likelihood of amyloid-related disease than non-carriers . TreatmeIn conclusion, this phase 3 trial was well conducted by the International Conference on Harmonization of Drug Development and Good Clinical Practice Guidelines. The trial demonstrated a robust and sustained drug-placebo difference on the primary outcome. We plan to conduct a global phase 3 trial to assess the safety and efficacy of GV-971 in additional populations. We will collect biomarkers in these trials and will interrogate the mechanism of action of GV-971 including its effects on neuroinflammation and gut dysbiosis.Additional file 1: Supplementary figures and tables.